Project Summary/Abstract The development of a vaccine capable of eliciting protective broadly neutralizing antibodies against HIV will require the development of improved strategies to control the antigenicity of envelope target antigens and adjuvants that can drive high levels of affinity maturation by eliciting sufficient/appropriate T-cell help, promoting germinal center development, and drive differentiation of long-lived plasma cells and memory B- cells to obtain a durable response. This P01 program focuses particularly on the membrane-proximal external region (MPER) of gp41 as a validated target for several broadly neutralizing antibodies, including the recently discovered 10E8 antibody that exhibits high potency without evidence of autoreactivity. In collaboration with the Reinherz lab (Project 1), we have recently developed a liposomal vaccine platform for delivery of hydrophobic, membrane-associated MPER fragments that allows durable, substantial titers against this poorly immunogenic peptide to be raised in normal mice. Building on this promising preliminary data and our very recent discoveries regarding the role of antigen oligomerization state on liposome surfaces in regulating B-cell triggering, we propose here a set of complementary strategies aiming to control the antigenicity and immunogenicity of MPER sequences, further enhance Tfh cell development and antibody affinity maturation, and promote humoral response durability. We will work in close collaboration with Project 1, providing liposomal vaccines and novel adjuvants on demand for their structure-focused studies, while pursuing a deeper understanding of how antigen presentation, adjuvant signaling, and T helper cell differentiation impact induction of high-affinity antibody responses against the MPER sequence of HIV. Our specific aims are (1) To determine the role of MPER oligomerization state on recognition by MPER-specific B-cells and humoral immunity, (2) To test strategies for controlling the angle of approach of antibodies to MPER sequences displayed on liposomal vectors, (3) To develop novel lymph node targeted adjuvants triggering the STING pathway for potent interferon-driven humoral responses, and (4) To test novel adjuvants directly modulating signaling pathways in helper T-cells to promote Tfh differentiation and germinal center induction. When combined with the structural advances pursued in Project 1, we aim to elicit broadly neutralizing antibodies by presenting MPER sequences in a correct configuration together with appropriate adjuvant signals that will promote the priming and affinity maturation of MPER-specific B-cells.